Using a single cell PCR method, we characterized the pathways that regulate B cell tolerance checkpoints by analyzing patients with primary immunodeficiencies. Alterations in B cell receptor signaling pathways in patients lacking functional btk, CD19, or molecules mediating TLR signaling such as IRAK-4 and MyDSS result in a defective central checkpoint and a failure to counterselect developing autoreactive B cells. In contrast, peripheral B cell tolerance requires CD40L/CD40 interactions and MHC class 11 expression, indicating that a CD4+ T cell population may regulate the removal of autoreactive B cells in the periphery. The decreased numbers of regulatory T (Treg) and CD4- natural killer T (NKT) cells as well as elevated B cell-activating factor (BAFF) levels in CD40L- and MHC class It-deficient patients suggest that these factors may contribute to the regulation of peripheral human B cell tolerance. The long range goal of the proposed research is to determine the mechanisms that regulate B cell tolerance in healthy humans but may be defective in patients with primary immunodeficiencies. The working hypothesis is that developing autoreactive B cells suffering from intrinsic genetic defects that alter BCR signaling threshold will fail to be counterselected in the bone marrow. In addition, non-functional Treg and CD4- NKT cells and/or elevated serum BAFF levels will interfere with peripheral B cell tolerance. In the proposed research, we will focus more specifically on the mechanisms by which developing autoreactive B cells are eliminated in the periphery by investigating FoxpS-deficient and X-linked lymphoproliferative disease (XLP) patients who lack Treg and NKT cells, respectively, and are likely to show a defective peripheral B cell tolerance checkpoint. In addition, mutations in either TACI or FAS genes associated with the development of common variable immunodeficiency disease (CVID) and autoimmune lymphoproliferative syndrome (ALPS), respectively, are expected to affect the removal of autoreactive B cells, therefore favoring the development of autoimmunity in these patients. RELEVANCE (See instructions): Understanding the mechanisms that prevent or account for the production of autoreactive B cells may suggest new approaches to control disease and design more specific and sustained therapies. In addition, anti-B cell therapies effective at blocking autoimmune disease progression may be useful for delaying or preventing the development of other disorders potentially affecting patients with primary immune deficiency.